The secretion of prolactin, a hormone that acts on the reproductive and immune systems, is regulated synergistically by dopamine and thyrotropin- releasing hormone (TRH). TRH and dopamine are known to affect tyrosine- and/or serine-threonine kinases, release of intracellular Ca2+, and electrical activity. However, the contribution of each of these actions to the control of prolactin secretion is unclear. Therefore, we have been using membrane capacitance measurements to follow exocytosis from single perforated patch clamped lactotrophs. Our results indicate that secretion is steeply dependent on Ca2+ influx. In addition, we have detected novel effects of TRH and dopamine on action potential activity. Furthermore, we have obtained evidence that protein phosphorylation might affect basal as well as stimulated secretion by maintaining and modulating voltage-gated Ca2+ channel activity. Finally, we have found that TRH acts in three phases to promote secretion under voltage clamp conditions. The multiple temporally distinct effects of TRH may be crucial for producing complex patterns of secretion that can be interactively controlled by dopamine. By combining membrane capacitance recordings with microfluorimetric detection of intracellular Ca2+, we propose to determine the roles of regulating action potential activity, voltage-gated Ca2+ channels, intracellular Ca2+ release and buffering, and the secretory apparatus in TRH-induced secretion. We will then determine if protein kinases and phosphatases are essential for TRH action. These studies will form the basis of further investigations on the mechanisms employed by dopamine to control basal and TRH-induced secretion. The proposed experiments will reveal how multiple modulators that activate distinct signal transduction mechanisms interact to control lactotroph secretory activity. It is likely that similar mechanisms will be utilized for coordinated regulation of peptide secretion by other endocrine cells and neurons.